Inbred corn line RBO1

ABSTRACT

An inbred corn line, designated RBO1, is disclosed. The invention relates to the seeds of inbred corn line RBO1, to the plants of inbred corn line RBO1 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line RBO1 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred RBO1.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line,designated RBO1. There are numerous steps in the development of anynovel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, and better agronomic quality.With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination and stand establishment, growthrate, maturity and plant and ear height is important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same corn traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new corn inbred line.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes. Thenew inbreds are crossed with other inbred lines and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificallyoften limit the usefulness of the approach.

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of grain produced on the land used and to supply food forboth animals and humans. To accomplish this goal, the corn breeder mustselect and develop corn plants that have the traits that result insuperior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred corn line,designated RBO1. This invention thus relates to the seeds of inbred cornline RBO1, to the plants of inbred corn line RBO1 and to methods forproducing a corn plant produced by crossing the inbred line RBO1 withitself or another corn line, and to methods for producing a corn plantcontaining in its genetic material one or more transgenes and to thetransgenic corn plants produced by that method. This invention alsorelates to methods for producing other inbred corn lines derived frominbred corn line RBO1 and to the inbred corn lines derived by the use ofthose methods. This invention further relates to hybrid corn seeds andplants produced by crossing the inbred line RBO1 with another corn line.

The inbred corn plant of the invention may further comprise, or have, acytoplasmic factor or other factor that is capable of conferring malesterility. Parts of the corn plant of the present invention are alsoprovided, such as e.g., pollen obtained from an inbred plant and anovule of the inbred plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture or inbred corn plant RBO1. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing inbred corn plant,and of regenerating plants having substantially the same genotype as theforegoing inbred corn plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

Another objective of the invention is to provide methods for producingother inbred corn plants derived from inbred corn line RBO1. Inbred cornlines derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a corn plantcontaining in its genetic material one or more transgenes and to thetransgenic corn plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of RBO1. The single transferred gene may preferably bea dominant or recessive allele. Preferably, the single transferred genewill confer such trait as male sterility, herbicide resistance, insectresistance, resistance for bacterial, fungal, or viral disease, malefertility, and enhanced nutritional quality. The single gene may be anaturally occurring maize gene or a transgene introduced through geneticengineering techniques.

The invention further provides methods for developing corn plant in acorn plant breeding program using plant breeding technique includingrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection and transformation. Seeds, corn plant, and parties thereofproduced by such breeding methods are also part of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

Predicted RM. This trait for a hybrid, predicted relative maturity (RM),is based on the harvest moisture of the grain. The relative maturityrating is based on a known set of checks and utilizes conventionalmaturity such as the Comparative Relative Maturity Rating System or itssimilar, the Minnesota Relative Maturity Rating System.

MN RM. This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Yield (Quintals/Hectare). The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

Moisture. The moisture is the actual percentage moisture of the grain atharvest.

GDU Silk. The GDU silk (=heat unit silk) is the number of growing degreeunits (GDU) or heat units required for an inbred line or hybrid to reachsilk emergence from the time of planting. Growing degree units arecalculated by the Barger Method, where the heat units for a 24-hourperiod are: GDU=((Max Temp+Min Temp)/2)−50 The highest maximum used is86 F and the lowest minimum used is 50 F. For each hybrid, it takes acertain number of GDUs to reach various stages of plant development.GDUs are a way of measuring plant maturity.

Stalk Lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Root Lodging. The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate 30angle or greater would be counted as root lodged.

Plant Height. This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Ear Height. The ear height is a measure from the ground to the ear nodeattachment, and is measured in centimeters.

Dropped Ears. This is a measure of the number of dropped ears per plot,and represents the percentage of plants that dropped an ear prior toharvest.

Stay Green. Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line RBO1 is a yellow dent corn with superiorcharacteristics, and provides an excellent parental line in crosses forproducing first generation (F₁) hybrid corn. Inbred corn line RBO1 isbest adapted to the areas of the United States corn belt with a latitudeof greater than 38 north and can be used to produce hybrids having arelative maturity of approximately 95-113 on the Comparative RelativeMaturity Rating System for harvest moisture of grain. Inbred corn lineRBO1 shows an excellent seedling vigor, an early pollen shed, excellentbrittle stalk resistance, superior root and stalk strength, and aboveaverage stay green.

RBO1 is similar to SBB1, however there are numerous differencesincluding the fact that RBO1 flowers slightly earlier than SBB1. Hybridswith RBO1 also are lower eared and have a higher test weight.

RBO1 has a plant height of 202 cm with an average ear insertion of 57cm. The kernels are arranged in distinct, slightly curved rows on theear. Heat units to 50% pollen shed are approximately 1412 and to 50%silk are approximately 1427.

RBO1 is an inbred line with very high yield potential and very strongstalk and roots in hybrids. For an inbred of its maturity, RBO1 resultsin a lower ear position in hybrid combination. Often these hybridcombinations results in plants which are of much better than averageoverall health when compared to inbred lines of similar maturity.

Some of the criteria used to select ears in various generations include:yield, stalk quality, root quality, disease tolerance, late plantgreenness, late season plant intactness, ear retention, ear height,pollen shedding ability, silking ability, and corn borer tolerance.During the development of the line, crosses were made to inbred testersfor the purpose of estimating the line's general and specific combiningability, and parallel evaluations were run in the USA by the Kirkland,Ill. Research Station. The inbred was evaluated further as a line and innumerous crosses by the Kirkland station and other research stationsacross the Corn Belt. The inbred has proven to have a good combiningability in hybrid combinations.

The inbred line has shown uniformity and stability for the traits,within the limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in RBO1.

Inbred corn line RBO1 has the following morphologic and othercharacteristics (based primarily on data collected at Kirkland, Ill.).

VARIETY DESCRIPTION INFORMATION

TYPE: Dent

REGION WHERE DEVELOPED: Northcentral Illinois.

MATURITY: 101 Days

Days Heat Units From emergence to 50% of plants in silk: 83 1427 Fromemergence to 50% of plants in pollen: 82 1412 Heat Units: = GDU = ((MaxTemp + Min Temp)/2) − 50

PLANT:

Plant Height to tassel tip: 201.7 cm (Standard Deviation=4.90)

Ear Height to base of top ear 56.9 cm (5.45)

Average Length of Top Ear Internode: 11.2 cm (0.26)

Average number of Tillers: 0.0 (0.00)

Average Number of Ears per Stalk: 1.0 (0.00)

Anthocyanin of Brace Roots: Absent

LEAF:

Width of Ear Node Leaf: 7.9 cm (0.16)

Length of Ear Node Leaf: 82.3 cm (0.96)

Number of leaves above top ear: 5.9 (0.57)

Leaf Angle (from 2nd Leaf above ear at anthesis to Stalk above leaf):75.8 (2.44)

Leaf Color: Dark Green Munsell Code 7.5 GY 4/4

Leaf Sheath Pubescence (Rate on scale from 1=none to 9=like peach fuz):8

Marginal Waves (Rate on scale from 1=none to 9=many): 8

Longitudinal Creases (Rate on scale from 1=none to 9=many): 7

TASSEL:

Number of Lateral Branches: 7.6 (2.01)

Branch Angle from Central Spike: 41.9 (1.85)

Tassel Length (from top leaf collar to tassel top): 46.1 cm (1.20)

Pollen Shed (Rate on scale from 0=male sterile to 9=heavy shed): 6

Anther Color: Light Purple to Pink Munsell Code 5 YR 4/2

Glume Color: Light Green Munsell Code 5GY 6/6

Bar Glumes: Absent

EAR: (Unhusked Data)

Silk Color (3 days after emergence): Greenish-Yellow Munsell Code 2.5 GY8/8

Fresh Husk Color (25 days after 50% silking): Medium Green Munsell Code5 GY 5/4

Dry Husk Color (65 days after 50% silking): Dark ta n Munsell Code 2.5GY 7/4

Position of Ear: Upright

Husk Tightness (Rate on scale from 1=very loose to 9=very tight): 4

Husk Extension at harvest: Short

EAR: (Husked Ear Data)

Ear Length: 13.5 cm (0.97)

Ear Diameter at mid-point: 41.4 mm (1.15)

Ear Weight: 111.6 gm (10.01)

Number of Kernel Rows: 15.4 (0.97)

Kernel Rows: Distinct

Row Alignment: Slightly Curved

Shank Length: 14.1 cm (3.73)

Ear Taper: Average

KERNEL: (Dried)

Kernel Length: 11.2 mm (0.51)

Kernel Width: 7.2 mm (0.43)

Kernel Thickness: 4.4 mm (0.51)

Round Kernels (Shape Grade): 43%

Aleurone Color Pattern: Homozygous

Aleurone Color: Colorless

Hard Endosperm Color: Yellow Munsell code 2.5Y 8/8

Endosperm Type: Normal Starch

Weight per 100 kernels (unsized sample): 25.1 gm (1.45)

COB:

Cob Diameter at Mid-Point: 23.7 mm (0.72)

Cob Color: Red Munsell code 10 R 4/10

AGRONOMIC TRAITS:

Stay Green (at 65 days after anthesis) (Rate on scale from 1=worst to9=excellent): 7

0% Dropped Ears (at 65 days after anthesis)

0% Pre-anthesis Brittle Snapping

0% Pre-anthesis Root Lodging

0% Post-anthesis Root Lodging (at 65 days after anthesis)

Yield of Inbred Per Se (at 12-13% grain moisture): 59 Bu/Acre

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of the line RBO1. Further, both first and second parent cornplants can come from the inbred corn line RBO1. Still further, thisinvention also is directed to methods for producing an inbred corn lineRBO1-derived corn plant by crossing inbred corn line RBO1 with a secondcorn plant and growing the progeny seed, and repeating the crossing andgrowing steps with the inbred corn line RBO1-derived plant from 0 to 7times. Thus, any such methods using the inbred corn line RBO1 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred corn lineRBO1 as a parent are within the scope of this invention, includingplants derived from inbred corn line RBO1. Advantageously, the inbredcorn line is used in crosses with other, different, corn inbreds toproduce first generation (F₁) corn hybrid seeds and plants with superiorcharacteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which corn plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, husks, stalks, roots, root tips, anthers, silk andthe like.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. P. Rao et al., Maize Genetics Cooperation Newsletter,60:64-65 (1986), refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987)indicates somatic embryogenesis from the tissue cultures of corn leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of corn is described in European Patent Application,publication 160,390, incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce corn plantshaving the physiological and morphological characteristics of inbredcorn line RBO1.

The utility of inbred corn line RBO1 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentiallysuitable for crosses with RBO1 may be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed corn plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Phisiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS, β-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incorn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incorn or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in corn.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin corn. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in corn. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application U.S.Ser. No. 93/06487, the contents of which are hereby incorporated byreference. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeusα-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract#497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, For Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content

1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127:87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

2) A gene could be introduced that reduced phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnisα-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentinvention, this also includes any single gene conversions of thatinbred. The term single gene converted plant as used herein refers tothose corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single gene of the recurrent inbred ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Industrial Applicability

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch inthe wet-milling industry and corn flour in the dry-milling industry. Theindustrial applications of corn starch and flour are based on functionalproperties, such as viscosity, film formation, adhesive properties, andability to suspend particles. The corn starch and flour have applicationin the paper and textile industries. Other industrial uses includeapplications in adhesives, building materials, foundry binders, laundrystarches, explosives, oil-well muds and other mining applications.

Plant parts other than the grain of corn are also used in industry, forexample: stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line RBO1, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

TABLES

In the tables that follow, the traits and characteristics of inbred cornline RBO1 are given in hybrid combination. The data collected on inbredcorn line RBO1 is presented for the key characteristics and traits. Thetables present yield test information about RBO1. RBO1 was tested inseveral hybrid combinations at numerous locations, with two or threereplications per location. Information about these hybrids, as comparedto several check hybrids, is presented.

The first pedigree listed in the comparison group is the hybridcontaining RBO1. Information for each pedigree includes:

1. Mean yield in Qx/Ha of the hybrid across all locations (Mean Yield)is shown in column 2.

2. A mean for the percentage moisture (% Moist) for the hybrid acrossall locations is shown in column 3.

3. A mean of the percentage of plants with stalk lodging (% Stalk)across all locations is shown in column 4.

4. A mean of the percentage of plants with root lodging (% Root) acrossall locations is shown in column 5.

5. A mean of the stay green across all location from 1 (poor) to 9(good) is shown in column 6.

6. Test weight is the grain density measured in kg per 100 liters isshown in column 7.

7. A mean of plant height in cm is shown in column 8.

TABLE I Overall Comparisons Hybrid vs. Check Hybrids Location: 2000 51Locations Mini-Strips Mean % % % Stay Test Plant Pedigree Yield MoistStalk Root Green Weight Height RBO1* 110.4 17.2 2.1 1.1 70.8 LH185 RBO1*108.8 18.4 1.3 0.9 69.5 LH287 At 51 Locations As Com- pared to: Pioneer107.9 17.3 4.5 1.8 72.1 Brand 35P12 Pioneer 105.4 17.6 2.2 6.8 73.4Brand 34G81

TABLE 2 Overall Comparisons Hybrid vs. Check Hybrids Location: 2000 29Locations UR8E01 Mean % % % Stay Test Plant Pedigree Yield Moist StalkRoot Green Weight Height RBO1* 93.8 19.4 3.8 1.4 5 69.4 275 LH185 RBO1*92.8 20.2 6.0 5.9 5 69.8 282 LH287 At 29 Locations As Com- pared to:Pioneer 92.1 20.0 6.9 9.2 6 70.3 280 Brand 35P12 Pioneer 88.8 19.0 11.015.8 4 72.0 293 Brand 34G81

TABLE 3 Overall Comparisons Hybrid vs. Check Hybrids Location: 1999 22Locations R8E02 Mean % % % Stay Test Plant Pedigree Yield Moist StalkRoot Green Weight Height RBO1* 101.6 19 1 0 6 71.0 268 LH185 At 15Locations As Com- pared to: Pioneer 98.1 18.9 2 1 6 74.4 277 Brand 34G81LH198* 94.7 19.7 1 1 7 71.5 252 LH 172

DEPOSIT INFORMATION

A deposit of the AgReliant Genetics corn inbred line RBO1 disclosedabove and recited in the appended claims has been made with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Virginia 20110. The date of deposit was Oct. 30, 2003. The deposit of2.500 seeds were taken from the same deposit maintained by AgReliantGenetics since prior to the filing date of this application. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. TheATCC accession number is PTA-5627. The deposit will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. An inbred corn seed designated RBO1, wherein asample of said seed has been deposited under ATCC Accession NumberPTA-5627.
 2. A corn plant, or parts thereof, produced by growing theseed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule orovules of the plant of claim
 2. 5. A corn plant, or parts thereof,having all of the physiological and morphological characteristics of thecorn plant of claim
 2. 6. The corn plant of claim 2, wherein said plantsaid plant further comprises a genetic factor conferring male sterlity.7. A tissue culture of regenerable cells from the corn plant of claim 2.8. A tissue culture according to claim 7, the cells or protoplasts ofsaid cells having been isolated from a tissue selected from the groupcosisting of leaves, pollen, embryos, roots root tip, anthers, silks,flowers, kerneles, ears, cobs, husks, and stalks.
 9. A corn plantregenerated from the tissue culture of claim 7, wherein the regeneratedplant expresses all the morphological and physiological characteristicsof inbred line RBO1, representative seed of said line having beendeposited under ATCC Accession No. PTA-5627.
 10. A corn plant with allof the physiological and morphological characteristics of corn inbredRBO1, representative seed of said inbred RBO1 having been depositedunder ATCC Accession No. PTA-5627, wherein said corn plant is producedby propagating said corn plant from the corn plant of claim 5 by atissue culture process.
 11. A method for producing a hybrid corn seedcomprising crossing a first inbred parent corn plant with a secondinbred parent corn plant and harvesting the resultant hybrid corn seed,wherein said first inbred parent corn plant or second said parent cornplant is the corn plant of claim
 2. 12. A method of producing atransgonic corn plant comprising transforming the corn plant of claim 2with a transgene wherein the transgene confers a characteristic selectedfrom the group consisting of: herbicide resistance, insect resistance,resistance to bacterial disease, resistance to fungal disease,resistance to viral disease, male sterility and wasty starch.
 13. Atransgenic corn plant produced by the method of claim
 12. 14. A methodof producing an herbicide resistant corn plant comprising transformingthe corn plant of claim 2 with a transgene that confers herbicideresistance.
 15. An herbicide resistant corn plant produced by the themethod of claim
 14. 16. A method of producing an insect resistant cornplant comprising transforming the corn plant of claim 2 with a transgenethat confers insect resistance.
 17. An insect resistant corn plantproduced by the method of claim
 16. 18. A method of producing a diseaseresistant corn plant comprising transforming the corn plant of claim 2with a transgene that confers disease resistance.
 19. A diseaseresistant corn plant produced by the method of claim
 18. 20. A method ofproducing a corn plant with decreased phytate content comprisingtransforming the corn plant of claim 2 with a tranagene encodingphytase.
 21. A corn plant with decreased phytate content, produced bythe method of claim
 20. 22. A method of producing a corn plant withmodified fatty acid or carbohydrate metabolism comprising transformingthe corn plant of claim 2 with one or more tranagenes encoding a proteinselected from the group consisting of stearyl-ACP desaturase,fructosyitransferase, levensucrase, aiphasmytase, invertase and starchbranching enzyme.
 23. A corn plant produced by the method of claim 22.24. A hybrid corn seed designated RBO1*LH185 having inbred line RBO1 asa parental line, representative seed having been deposited under ATCCAccession No PTA-5627 and inbred line LH185, representative seed havingbeen deposited under ATCC Accession No.
 75618. 25. A hybrid corn seeddesignated RBO1*LH287 having inbred line RBO1 as a parental line,representative seed having been deposited under ATCC Accession NoPTA-5627 and inbred line LH287, representative seed having beendeposited under ATCC Accession No. PTA-1174.
 26. A method of introducinga desired trait into corn inbred line RBO1 comprising: (a) crossing theRBO1 plants, grown from seed deposited under ATCC Accession No.PTA-5627, with plants of another corn line that comprise a desired traitto produce F1 progeny plants, wherein the desired trait is selected frommale sterility, herbicide resistance, insect resistance, waxy starch andresistance to bacterial disease, fungal or disease viral disease; (b)selecting F1 progeny plants that have the desired trait to produceselected F1 progeny plants; (c) crossing the selected F1 progeny plantswith the RBO1 plants to produce first backcross progeny plants; (d)selecting for first backcross progeny plants that have the desired traitand physiological and morphological characteristics of maize inbred lineRBO1 listed in the Variety Description Information to produce selected[first] backcross progeny plants; and (e) repeating steps (c) and (d)three or more times in succesion to produce selected fourth or higherbackcross progeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of maize inbred RBO1 asdetermined at a 5% significance level when grown in the sameenvironmental conditions.
 27. A plant produced by the method of claim26, wherein the plant has the desired trait and all of the physiologicaland morphological characteristics of corn inbred line RBO1 as determinedat a 5% signicance level when grown in the same environmentalconditions.
 28. A corn plant produced by growing the corn seed of claim24.
 29. A method comprising crossing the corn plant of claim 24 withitself or another corn plant to produce a seed.
 30. A corn plantproduced by growing the corn seed of claim
 25. 31. A method comprisingcrossing the corn plant of claim 25 with itself or another corn plant toproduce a seed.